The Chlamydia genus contains members representing significant health concerns in the United States. A significant burden exists due to prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular parasites that undergo a complex developmental cycle that is accompanied by distinct alterations in gene expression, morphology, and protein function. For example, expression and activity of the virulence-associated type III secretion system (T3SS) is intimately coupled to developmental progression. In addition, disulfide bond dynamics in the outer envelope of Chlamydia spp. has a dominant, yet incompletely understood, role in the developmental cycle. We have preliminary evidence that T3S proteins participate in developmentally associated alterations in disulfide bonding. We propose a combination of methods designed to characterize disulfide bonding among T3S apparatus proteins to establish the extent, temporal dynamics, and impact on secretion activity. A great deal of emphasis has been placed on identification and characterization of secreted effector proteins while comparatively little is known regarding more direct contributions of the T3S apparatus to chlamydial biology. Although the T3S process displays a significant degree of functional conservation among Gram-negative pathogens, it is likely that the highly unique niche colonized by chlamydiae results in mechanisms novel to the chlamydial system. Therefore, these studies will add a new dimension to knowledge regarding the T3S mechanism, lead to an enhanced understanding of Chlamydia-mediated disease, and potentially yield novel preventative and treatment therapies.